Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via complex intracellular routes. Though diverse in their intracellular targets, a common and essential step in their virulence is the ability to reach the cytosol, where most toxins exert their enzymatic effects. The bacterial products Stx and Ctx, as well as the plant toxin ricin, traffick in a retrograde fashion from endosomes to the endoplasmic reticulum via the Golgi while bypassing lysosomal degradation. It has become increasingly apparent that this retrograde trafficking pathway is not unique to bacterial toxins. Rather, bacterial toxins are believed to hijack existing host transport pathways. The sequential retrograde progression utilized by these toxins has translated into a unique system for probing host endocytic mechanisms and have enhanced our understanding of retrograde transport. In an effort to dissect and inhibit the stepwise trafficking of Stx, we have developed an alternative approach toward inhibiting toxin transport. Previous genetic approaches aimed at identifying components essential to toxin trafficking have proven inconsistent and revealed the complexity underlying these pathways. As a result, we have applied a highly sensitive, high-throughput luciferase-based assay to screen a library of small molecule compounds for their ability to block inhibition of protein synthesis. From an initial screen, we have identified various compounds that strongly protect against Shiga intoxication. One novel compound, B06, produces a brefeldin A-like dispersal of the Golgi, and preliminary evidence suggests that it could serve as a useful tool in elucidating ARF1-mediated transport through the Golgi apparatus. In addition, we hope to explore the role of kinases in toxin trafficking. Though there is evidence for the role of kinases in toxin endocytosis, their functions in intracellular toxin trafficking remain unclear. A novel kinome siRNA library will be screened by the luciferase-based assay to identify kinases essential to Stx transport. More importantly, the identification of kinases involved at various stages along the Stx trafficking pathway will further our understanding of their functions in mediating intracellular transport. Identification of kinases involved at distinct steps in toxin trafficking will equally provide therapeutic possibilities against toxin-mediated diseases. Bacterial and plant toxins are significant agents of human disease and potential vehicles for bioterrorism. Our high-throughput assay underscores the therapeutic utility of a small molecule approach for targeting toxin-mediated diseases.